Qiuxi Ding ,Ruoyu Zh ,Xinye He ,Ao Sun ,Kun Shi ,Ying Qu ,Zhiyong Qin,*

a State Key Laboratory of Biotherapy and Cancer Center,West China Hospital,West China Medical School,Sichuan University,Chengdu,610041,China

b Urology Institute of Shenzhen University,The Third Affiliated Hospital of Shenzhen University,Shenzhen University,Shenzhen,518000,China

Keywords:Porous PDLLA microspheres Fibroblast Collagen Soft-tissue augmentation

ABSTRACT This study designed a novel porous PDLLA microspheres/collagen gel composite combined with fibroblasts as the injectable dermal filler for soft-tissue augmentation.The low degradation rate of porous PDLLA microspheres and seed cells were introduced in the system to achieve the filling volume stability and stronger regeneration activity.Biodegradable PDLLA porous microspheres were prepared by an improved water-in-oil-in-water double emulsion solvent evaporation method.The combine of fibroblasts with porous microspheres was observed through co-culture of cells with microspheres,SEM (scanning electron microscope) and fluorescent staining technology.The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,the hemolysis test and histocompatibility experiment indicated that the composite had satisfactory biocompatibility in vitro and in vivo.In the animal studies,the composite was implanted into the subcutaneous part of the rats’ back through sterile needles.The collagen was metabolized within 30 days,meanwhile,fibroblasts-porous PDLLA microspheres served as scaffold structure and seed cells which promote new connective tissue formation.The histopathological studies demonstrate that the fibroblasts-porous PDLLA microspheres/collagen gel composite group could create larger amount of supportive structure than PDLLA porous microspheres/collagen gel composite group.All the results indicate that fibroblasts-porous PDLLA microspheres/collagen gel composite may have great clinical application in soft-tissue augmentation with its excellent biocompatibility,regeneration activity and stable long-term filling ability.*Corresponding author.

1.Introduction

Soft-tissue augmentation is a process of implanting tissues,cells or materials to dermal or subcutaneous tissues for aesthetic reasons or supporting purposes with the advantages of a low risk of complications and a short recovery time [1].Currently,the available injectable softtissue augmentations can be classified into three categories:replacement augmentations(collagen,hyaluronic acid)[2–4],semi-permanent augmentations (degradable biomaterials) and permanent augmentations (polymethylmethacrylate,silicone and hydroxy-ethylmethacrylate) [5].The use of replacement augmentations is limited because of their short duration periods of 3–6 months.The permanent augmentations can be expected to produce long-term results with a single injection.However,the remedies could be problematic when imperfections in treatment take place which often demand re-operation [6,7].With careful assessment,semi-permanent augmentations of degradable biomaterials are the most ideal augmentations in the current clinical environment [8].Clinically,the emergence of soft tissue augmentation materials which meet safety,effectivity,and practicality is urgently expected.

The ideal biologically active soft tissue filling material refers to a function of not only participate in normal physiological metabolism in the body but also secreting various growth factors and other functional proteins which could promote the formation of new tissue.Therefore,a tissue augmentation biomaterials should contain physiological active cells.Currently involved in the field of orthopedics are mainly fibroblast transplantation [9] and stem cells [10–12].The first-in-class personalized cell therapy for eliminating finfa wrinkles or nasolabial folds around the nose and mouth was approved by the US FDA in 2011,fibroblasts are thought to produce collagen and other growth factors to form the matrix under skin [13,14].However,in addition to the seed cells,we also need a similar structure to the extra-cellular matrix to provide an attachment point for cell growth and reproduction,as well as the transport of nutrients.

Biodegradable polymeric biomaterials have been widely investigated as a structural supporting matrix for tissue regeneration,drug delivery and recycle waste batteries [15–20].In this study,a new model of porous PDLLA microspheres were prepared by a modified water-in-oil-in-water (w/o/w) double emulsion solvent evaporation method and used as extra-cellular matrix for fibroblasts attachment and proliferation which can not only provide a good anchor for cell adsorption,but also facilitate the transport of nutrients and oxygen for the seed cells.

Collagen is the principal structural element of the extra-cellular matrix [21,22].The breaks down of collagen followed with some growth factors and cytokines which generate new neocollagenesis and neovascularization.While collagen also has some disadvantages,one of the concerns is the longevity;collagen will undergo rapid proteolytic degradation.To increase the longevity,intermolecular or intramolecular cross-linking of collagen through physical treatments or chemical agents are utilized,which prevents rapid degradation.How to solve the life of collagen is an important direction of current research[23,24].In our study,collagen could provide support and nutrition to seed cells,and then fibroblasts-porous PDLLA microspheres can promote collagen regeneration which could compensate for the rapid degradation of collagen.On the other hand,collagen gel served as a vehicle for subcutaneous injection of fibroblasts-porous PDLLA microspheres and had good thermosensitivity.

As shown in schematic 1,an injectable fibroblasts-porous PDLLA microspheres/collagen gel composite was prepared for soft-tissue augmentation.The composite combined the advantages of porous PDLLA microspheres,fibroblasts and thermosensitive collagen gel.The characterization of porous PDLLA microspheres suggested that it could be excellent similar structure to the extra-cellular matrix.Fibroblasts attachment and proliferation on porous PDLLA microspheres were observed using SEM and LSCM (laser scanning confocal microscope).The animal studies demonstrate that the abilities of this novel composite for stimulating collagen regeneration and creating supportive structure were excellent.All results suggest that the fibroblasts-porous PDLLA microspheres/collagen gel composite may be an effective and promising biomaterial for soft tissue augmentation.

2.Experimental procedure

2.1.Materials

PDLLA was purchased from NatureWorks (USA,4032D,Mn=135,000).Polyvinylalcohol(PVA,Av,Mn=30,000–70,000)was purchased from Sigma.Methylene chloride,ammonium bicarbonate,sodium hydroxide and phosphate buffer saline were purchased from Kelong Chemicals,Chengdu,China.All chemicals were of analytic reagent (AR) grade and used directly without further purification.Collagen was purchased from Mingrang Biological Science-Technology Company,Chengdu,China.Ultrafiltrated water was obtained from Milli Q plus,Millipore (USA).Dulbecco's modified Eagle's medium (DMEM,Sigma,USA)contained 10%fetal bovine serum(FBS,Gibico BRL,USA)was used without any treatment.

SD (Sprague-Dawley) rats weighing 230 ± 20 g were purchased from Laboratory Animal Center of Sichuan University.Animals were housed in an animal facility of SPF class.They were kept at controlled temperature range of 20–22 °C,50–60% relative humidity and 12 h of light-dark cycles.Free access to food and water was allowed.All animals were kept in quarantine for at least a week before treatment.

2.2.Preparation and characterization of fibroblasts-porous PDLLA microspheres/collagen gel composite

2.2.1.Isolation and culture of fibroblasts

Fibroblasts were obtained from the corium of the newborn SD rat under sterile conditions and minced into small pieces.After digestion with 1 mg/ml collagenase type I for 5 h,the fibroblasts were collected and cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS).

2.2.2.Preparation of porous PDLLA microspheres

PDLLA porous microspheres were prepared by a modified water-inoil-in-water (w/o/w) double emulsion method.Briefly,0.6 g PDLLA was dissolved in 20 ml methylene chloride.Following,6.25 ml of deionized water containing 10%NH4HCO3was added into the solution and stirred using a homogenizer at 5000 rpm for 3 min in an ice bath.The primary emulsion was immediately poured into a beaker containing 750 ml of 0.1% (w/v) PVA solution and re-emulsified at 500 rpm for 6 h at room temperature.The PDLLA porous microspheres were soaked into NaOH aqueous solution and stirred for 15–20 min.The solidified microspheres were washed 5 times with deionized water and lyophilized using a freeze dryer.

2.2.3.Preparation and characterization of collagen gel

0.35 g collagen was dissolved in 10 ml physiological saline at room temperature.The collagen solution was heated in a 60 °C water bath.Then,the collagen solution was cooled to 37 °C and the collagen gel with concentration of 3.5% (w/v) was prepared successfully.

2.2.4.Preparation of fibroblasts-porous PDLLA microspheres/collagen gel composite

The collagen solution is prepared and cooled to 39 °C.0.054 g fibroblasts-porous PDLLA microspheres were centrifuged at 1500 rpm for 3min after 7 days cultivation,and then resuspended with collagen solution (39 °C).The fibroblasts-porous PDLLA microspheres/collagen solution was cooled to 37 °C immediately and the fibroblasts-porous PDLLA microspheres/collagen composite was prepared.

2.3.Morphologic observation

The surface morphology of porous PDLLA microspheres,collagen thermosensitive hydrogel,fibroblasts-porous PDLLA microspheres/collagen gel composite was observed using scanning electron microscope(SEM,JEM-100CX,Japan).The microspheres were coated with gold using a sputter coater (KYKY SBC-12,China) and analyzed at 20 kV.The diameter and pore size of porous PDLLA microspheres were determined by calculating from SEM micrographs.Their averages and standard deviations were investigated.

2.4.Porosity and bet surface area

The porosity and bet surface area of porous PDLLA microspheres were measured using a Quanta chrome Autoscan-33 mercury intrusion porosimeter with capable of a maximum pressure of 33,000 psia(227 MPa).

2.5.Rheological study

Rheological measurement of the collagen thermosensitive hydrogel and fibroblasts-porous PDLLA microspheres/collagen gel composite was performed using an AR2000 ex rheometer (TA Instruments,USA).The samples were put between parallel plates of 40 nm diameter and with a gap of 1 mm.The cooling rate was 2°C/min in the range 60–10 °C.The storage modulus (G′),loss modulus (G″) and complex viscosity (n＊) were measured as functions of temperature.

2.6.Collagen analysis

At preset intervals,fibroblasts-porous PDLLA microspheres were trypsinized,washed 3 times with PBS and lyophilized using a freeze dryer.The sample was digested in papain solution at 60°C for 16 h.The papain-digested sample was added with 6 N HCl and autoclaved at 121 °C for 20 min for hydrolysis.Samples were neutralized by adding sodium hydroxide solutions,diluted with deionized water,and added with chloramine-T solution.Then,p-dimethylaminobenzaldehyde was added,incubated at 60 °C,cooled and the absorbance value was measured at 550 nm using L-hydroxyproline as a standard [25].

2.7.In vitro and in vivo degradation of porous PDLLA microspheres and collagen gel

For in vitro degradation of porous PDLLA microspheres,microspheres (0.6 mg) were immersed in phosphate buffer solution (PBS,8 ml,0.1 M,pH 7.4)on an orbital shaker at 37°C with a shaking speed of 50 rpm.The buffer solution was renewed every three day.At specific time intervals,the samples were removed from the buffer solution and lyophilized using a freeze dryer.The macromolecular weight and weight distribution of PDLLA copolymer were determined using GPC(Agilent 110 HPLC,USA).

SD rats were used to evaluate the in vivo degradation of PDLLA porous microspheres.Microspheres were sterilized by Cobalt-60 irradiation.0.6 mg of sterilized microspheres were suspended in 0.5 ml of physiological saline and injected into the back of SD rats subcutaneously under sterile conditions.At preset intervals,three rats were sacrificed by overdose anesthetics.The microspheres with excess surrounding tissue were removed from the injection site and homogenized with tissue homogenizer (Ultra-Turrax T 10 basic,IKA,Germany) for 1 min.The samples were lyophilized and the microspheres were isolated from them.The macromolecular weight and macromolecular weight distribution of PDLLA copolymer were determined through GPC(Agilent 110 HPLC,USA).

0.5 ml collagen gel with concentration of 3.5%(w/v)was sterilized by Cobalt-60 irradiation.SD rats were anesthetized and the collagen gel subcutaneously injected into them.At predetermined time,the SD rats were sacrificed.The degradation process of collagen gel was observed.

2.8.Cell attachment and proliferation analysis

Cell attachment and proliferation on porous PDLLA microspheres were observed using SEM and Laser scanning confocal microscope(LSCM,Leica,Germany).24 h after the cultivation,fibroblasts-porous PDLLA microspheres were fixed with 2.5% glutaraldehyde in PBS for 4 h,dehydrated in a graded series of ethanol/water solution,dried and sputter-coated with gold-palladium (AuPd),and finally observed on a SEM.3 days after the cultivation,the fibroblasts-PDLLA porous microspheres were rinsed with PBS twice,and then stained with 0.1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) for 5 min.Subsequently,fibroblasts-porous PDLLA microspheres were washed 3 times with PBS and observed using a LSCM.

2.9.Cytotoxicity assay

Cytotoxicity of fibroblasts-porous PDLLA microspheres/collagen composite to fibroblasts was evaluated by MTT assay according to ISO 10993-5 guideline.The composite was extracted using DMEM for 24 h.The fibroblasts were incubated with the composite leachates of different concentration(25%,50%,100%,n=3)up to 6 days.Then,80 μl of MTT was added to each well and the fibroblasts were further incubated for another 4 h.The precipitated formazan was dissolved in 640 μl DMSO and the absorbance at 570 nm was measured.We repeated the experiments in five independent experiments.

2.10.In vitro hemolysis study

Fig.1.A,A-1:SEM images of porous PDLLA microspheres.B,B-1,C,C-1:SEM photographs of collagen gel surface (B/B-1) and cross-section (C/C-1).D,D-1:SEM photographs of porous PDLLA microspheres/collagen gel composite surface (D/D-1) and cross-section (E/E-1).

The hemolysis study was performed on fibroblasts-porous PDLLA microspheres/collagen composite in normal saline solution with collagen concentrations ranging from 2 to 40 mg/ml.Briefly,0.5 ml of the composite (13 wt% of the composite were fibroblasts-porous PDLLA microspheres) with different collagen concentrations was diluted into 2.5 ml by normal saline and added into 2.5 ml of rabbit erythrocyte suspension (2%) in normal saline at 37 °C.A normal saline negative control and distilled water positive control were also detected for comparison.The erythrocyte suspension was centrifuged and the supernatant color was compared with controls 3 h later.The collected supernatants were detected on a UV–Vis spectrophotometer at 545 nm to test the hemolytic rate.The hemolytic rate was calculated according to the following equation:

Fig.2.Confocal laser scanning microscope results of fibroblasts on porous PDLLA microspheres for 5 days (A:1 day,B:three days,C:5 days).SEM photographs of fibroblasts cultured on porous PDLLA microspheres (D,E).

2.11.In vivo persistence of fibroblasts-porous PDLLA microspheres/collagen gel composite

Fibroblasts-porous PDLLA microspheres/collagen gel composite,porous PDLLA microspheres/collagen gel composite and fibroblasts was subcutaneously injected into the back of male SD rats.At specific time intervals,3 rats were sacrificed.The injection sites were carefully opened and then,the photographs were taken.The height,width and length of the composite was measured with digital calipers (Digimatic calipers,Mitutoyo America,Aurora,IL) and the volumes of the composite was calculated.The volume was calculated according to the following equation:

2.12.In vivo histological examination

0.5 ml of fibroblasts-porous PDLLA microspheres/collagen gel composite was subcutaneously injected into Male SD rats.7,60,120 and 180 days after injection,the fibroblasts-porous PDLLA microspheres/collagen gel composite along with their surrounding tissues were carefully collected and fixed with 10%neutral formalin for 2 days.Subsequently,the samples were embedded in paraffin,sawed using a microtome (Leica SpA,Milan,Italy) and stained with H&E and Trichrome-Masson to determine the composite biocompatibility and ability for stimulating collagen regeneration.The number of inflammatory cells was quantified with a computer-based image analysis system (Image-Pro Plus,Silver Spring,MD,USA).

2.13.Statistical analysis

All quantitative data were expressed as a mean ± standard deviation.Statistical analysis was performed by SPSS software.A value of P ＜0.05 was considered statistically significant.

3.Results and discussion

3.1.Characterization of fibroblasts-porous PDLLA microspheres/collagen gel composite

Fig.1 showed SEM images of porous PDLLA microspheres which were spherical in shape and had a highly open-pore structure.The diameter and pore size of porous PDLLA microspheres were 328 ± 39 μm and 12 ± 6 μm respectively.The porosity and bet surface area of porous microspheres were measured by mercury intrusion porosimetry.The results demonstrated that the porosity and bet surface area were 81.31 ± 0.87% and 230.00 ± 0.56 m2/g respectively.Fig.1 B–C presented the appearance of the collagen gel.The collagen gel presented three-dimensional network structure.The SEM photographs of fibroblasts-porous PDLLA microspheres/collagen gel composite was showed in Fig.1D and E.The data demonstrated that porous PDLLA microspheres were distributed uniformly in the threedimensional network structure of collagen gel.

Fig.3.Confocal laser scanning microscope images of porous PDLLA microspheres after fibroblasts cultivation for 5 days.

Fig.4.The extracellular matrix deposition was assessed by measuring the collagen content(μg)by the hydroxyproline assay.In vitro culture of fibroblasts cultured on solid PDLLA microspheres (monolayer) and on porous PDLLA microspheres for 4 weeks.

Fig.5.The appearance of fibroblasts-porous PDLLA microspheres/collagen gel composite (A,B).Rheological results showing changes of storage modulus (G′),loss modulus(G″),and complex viscosity(n＊)of samples upon heating process.C:collagen gel,D:fibroblasts-porous PDLLA microspheres/collagen gel composite.In vivo degradation of collagen.Legend:arrows-the remaining collagen.

As shown in Fig.2,microspheres could give fibroblasts a good environment for adhesion.Fibroblasts were adhered on the surface of microspheres and also presented within the pores of the microspheres after one day cultivation.Fig.3 showed confocal images of fibroblasts with the nucleus stained by DAPI after being seeded onto microspheres.It is demonstrated that fibroblasts can proliferate during 5 days cultivation.Fig.4 showed the results of collagen content during 4 days cultivation.The amine-functionalized porous PDLLA microspheres showed superior capacity of depositing ECM components during cultivation than others.

From Fig.5A and B,the pictures demonstrated the appearance of fibroblasts-porous PDLLA microspheres/collagen gel composite.It is in a flowing state at a higher temperature and is in a solidified state at 37°.As Fig.5C and D showed the result of rheological study,the change of aqueous samples from a liquid-like to an elastic gel-like state occurred at the crossover point of G′and G″,which was observed at 38°C as the test temperature changed from 60 to 10 °C.The results declared that fibroblasts-porous PDLLA microspheres/collagen gel composite had obvious thermal sensitivity.Fig.5E–G presented the photograghs taken at 1st day,14th day and 30th day after collagen gel subcutaneously injected into SD rats.The size of gel decreased with the time going on and the gel was metabolized completely at 30th day.

In tissue engineering,a high porosity (often ＞80%) is desired for microcarriers to provide sufficient space for cell growth and ECM deposition [26–28].A high surface area probably enhances protein adsorption for cell-microcarriers interactions and facilitates mass transfer for tissue regeneration [29,30].The properties of porous PDLLA microspheres suggested that it could be excellent microcarrier.The in vitro attachment and proliferation of fibroblasts to porous PDLLA microspheres were examined and the results affirmed the ability of microspheres to be the microcarrier for fibroblasts.The pores of 12 ± 6 μm in diameter allow the seeded fibroblasts to infiltrate into the interior region of well-interconnected porous PDLLA microspheres.

Fig.6.Fig.6 The cell viability of porous PDLLA microspheres/collagen gel composite.Pourous PDLLA microspheres;Pourous PDLLA microspheres/collagen gel composite (25%);Pourous PDLLA microspheres/collagen gel composite (50%);Pourous PDLLA microspheres/collagen gel composite (100%).

In this research,collagen gel served as a vehicle for subcutaneous injection of fibroblasts-porous PDLLA microspheres and had good thermosensitivity.Collagen solution underwent gel-sol transition as the temperature changed from 60 °C to 10 °C,fibroblasts could have some damage at 39 °C.However,they could be restored after a little while because of the tolerable temperature and the short time of high temperature[31].The fibroblasts-porous PDLLA microspheres/collagen gel composite had the same thermosensitivity as the collagen gel.The three-dimensional network structure of collagen gel is desirable to enhance mass transports of oxygen and nutrients for fibroblasts-porous PDLLA microspheres.The collagen gel was metabolized within 30 days in vivo.At the same time,each single microsphere was encapsulated with collagen fibers secreted by the fibroblasts.

3.2.Compatibility and biodegradability evaluation of porous PDLLA microspheres in vivo and vitro

As the result of MTT assay depicted in Fig.6,fibroblasts proliferated in all composite.The cell viability of fibroblasts was obviously higher in porous PDLLA microspheres/collagen gel composite than pure porous PDLLA microspheres group.

Fig.7A-E is the result of the organ histocompatibility treated with fibroblasts-porous PDLLA microspheres/collagen gel composite.The tissue cells from organs were clear and arranged in good order,and no necrosis,hemorrhage and inflammatory exudates were observed.

The hemocompatibility of the fibroblasts-porous PDLLA microspheres/collagen gel composite was studied.As shown in Fig.7F of hemolytic test,fibroblasts-porous PDLLA microspheres/collagen solution with collagen concentrations ranging from 2 to 40 mg/ml did not cause any hemolysis on rabbit erythrocytes.The hemolytic rate was lower,which implied that the composite had great hemocompatibility.

GPC measurements were carried out to study the changes of the Mn,Mw and Mw/Mn during degradation in vitro and in vivo.As shown in Table 1,the porous PDLLA microspheres degraded slowly both in vitro and in vivo.In vivo,the Mn and Mw decreased gradually to 52% and 61%of the initial value after 150 days.In vitro in PBS(PH 7.4),the Mn and Mw decreased gradually to 60% and 66% of the initial value after 150 days.The polydispersity (Mw/Mn) increased along with degradation,both in vitro and in vivo.

Table 1 In vitro and in vivo degradation of porous PDLLA microspheres.

The biocompatibility of the fibroblasts-porous PDLLA microspheres/collagen gel composite was studied both in vitro and in vivo.The results demonstrate that the composite was safe with good biocompatible.The biodegradability of composite can be safely used in vivo.

3.3.Soft tissue filling effect of fibroblasts-porous PDLLA microspheres/collagen gel composite

A subcutaneous rat model was used instead of a larger animal dermal model because of its efficiency and affordability.Fibroblastsporous PDLLA microspheres/collagen gel composite and porous PDLLA microspheres/collagen gel composite was injected into the back of the rat for creating supportive structure and foundation.As displayed in Fig.8A and B,both groups could be skin augmentation.The persistence size of porous PDLLA microspheres/collagen gel composite in SD rat skin have a more dramatic decline from 1st day to 30th day compared with fibroblasts-porous PDLLA microspheres/collagen gel composite and have a slow decline from 30th day to 180th day.However,the persistence size of fibroblasts-porous PDLLA microspheres/collagen gel composite nearly remained unchanged from 30th day to 180th day.

Fig.7.Light micrographs of normal tissues in rat treated with fibroblasts-porous PDLLA microspheres/collagen gel composite(×200):rat cardiac muscle(A),liver(B),spleen(C),lung (D),and kidneys (E).Hemolytic tests of fibroblasts-porous PDLLA microspheres/collagen gel composite (F).The collagen concentration was 1:2 mg/ml;2:5 mg/ml;3:10 mg/ml;4:20 mg/ml;5:40 mg/ml;sample 6 was normal saline used as negative control,sample 7 was distilled water used as positive control.

The results for volumetric validation were showed in Fig.8C.After the initial injection on day 1,the persistence volumes of fibroblastsporous PDLLA microspheres/collagen gel composite,porous PDLLA microspheres/collagen gel composite was 224.2 ± 90.6 mm3,219.7 ± 72.1 mm3,respectively.7 days after the injection,the persistence volumes of fibroblasts-porous PDLLA microspheres/collagen gel composite,porous PDLLA microspheres/collagen gel composite was 253.4 ± 80.4 mm3,224.8 ± 91.3 mm3,respectively.30 days after the injection,the persistence volumes of fibroblasts-porous PDLLA microspheres/collagen gel composite,porous PDLLA microspheres/collagen gel composite and fibroblasts changed to 224.8 ± 71.0 mm3,132.3 ± 30.1 mm3,respectively.The filling volume of the two groups has obvious differences.

The apparent decrease in the filling volume of the two groups in the first 30 days was mainly due to the degradation of collagen.However,fibroblasts can promote collagen regeneration and they also can proliferate,which could reduce volume changes.Therefore,the decrease in the filling volume of fibroblasts-porous PDLLA microspheres/collagen gel composite is lower than control group.Fibroblasts-porous PDLLA microspheres/collagen gel composite is a better soft tissue filling material compared with no seed cells.

3.4.Histological examination of soft tissue augmentation in vivo

Fig.8.In vivo porous PDLLA microspheres/collagen gel composite (A),fibroblasts-porous PDLLA microspheres/collagen gel composite and fibroblasts persistence(B).Images were taken at 1 day,30 days,120 days and 180 days after subcutaneous injection of samples into the back of the SD rats.Legend:arrows-the remaining samples.The changes of the filling volume of porous PDLLA microspheres/collagen gel composite and fibroblasts-porous PDLLA microspheres/collagen gel composite (C).

Fig.9.Soft tissue reaction to fibroblasts-porous PDLLA microspheres/collagen gel composite.Optical micrographs were taken of H&E-stained histology slides at × 100 and × 400.The micrographs were taken of fibroblasts-porous PDLLA microspheres/collagen gel composite that had been injected for(A),(A-1):7 days;(B),(B-1):30 days;(C),(C-1):120 days;(D),(D-1):180 days.

Histological examination of soft tissue augmentation was studied by HE and Masson stained.As shown in Fig.9,a slight inflammatory reaction occurred at 7th day.It can be seen that the inside of the microspheres have some cells and no tissues,but as time progresses,in the interior of the fibroblasts-porous PDLLA microsphere,the tissues grow gradually.

The capacity for stimulating collagen regeneration by fibroblastsporous PDLLA microspheres/collagen gel composite was assessed through histological section observation.As seen in Fig.10,the density of collagen fibers in the interior of the fibroblasts-porous PDLLA microspheres gradually increased from 7th day to 180th day.In 7th day,the collagen existed mainly out of the fibroblasts-porous PDLLA microspheres/collagen gel composite.At 180th days,the interior of fibroblasts-porous PDLLA microspheres/collagen gel composite was totally filled with collagen.In summary,the injectable fibroblasts-porous PDLLA microspheres/collagen gel composite is a good candidate and superb clinical application prospects for skin augmentation.

Fig.10.Soft tissue reaction to fibroblasts-porous PDLLA microspheres/collagen gel composite.Optical micrographs were taken of Trichrome-Massonstained histology slides at × 100 and × 400.The micrographs were taken of fibroblasts-porous PDLLA microspheres/collagen gel composite that had been injected for (A),(A-1):7 days;(B),(B-1):30 days;(C),(C-1):120 days;(D),(D-1):180 days.

The ideal filler should be long-lasting,biocompatible,chemically inert,soft and easy to use,and have a long history of safety.Over the years,a number of injectable materials have evolved in the treatment of soft tissue defects,including polymers and cell populations.In Chang's study [32],the levan hydrogel with injectability,biocompatibility,and an anti-wrinkle effect,however,this composite has a fast degradation rate and not suitable for long-term filling.Most fillers are just a simple filling function,and cannot promote cell regeneration well[33,34].The excellent performance of fibroblasts-porous PDLLA microspheres/collagen gel composite could stimulate collagen regeneration,the longterm and continuous filling capacity.Fibroblasts are thought to produce collagen and other growth factors to form the matrix under skin.The porous PDLLA microspheres act as the extra-cellular matrix to provide an attachment point for cell growth and reproduction with good biocompatibility and easy to use.

4.Conclusion

Fibroblasts-porous PDLLA microspheres/collagen gel composite has been prepared as skin augmentation to increase the corium thickness.Highly open porous PDLLA microspheres have been produced by a modified water-in-oil-in-water (w/o/w) double emulsion method.The resultant porous biodegradable microspheres can be used as temporal scaffold microcarriers for attachment and proliferation of fibroblasts.The fibroblasts-containing microcarriers (fibroblasts-porous PDLLA microspheres) suspended in collagen gel to fabricate fibroblasts-porous PDLLA microspheres/collagen gel composite.The composite has excellent biocompatibility and thermosensitivity.The histopathological studies indicat that the composite can significantly stimulate collagen regeneration.After the fibroblasts-porous PDLLA microspheres/collagen gel composite is injected subcutaneously,the volume of the composite approximately remains constant in the experimental process.All the results suggest that fibroblasts-porous PDLLA microspheres/collagen gel composite may present a fair future for skin augmentation.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by The National Natural Science Foundation of China (NSFC31525009,81502690),National Key Research and Development Program of China (No.2017YFC1103502,2017YFC1103500),and 1·3·5 project for disciplines of excellence,West China Hospital,Sichuan University (ZYGD18002).